Characterization of the α-helix region in domain 3 of the haemolytic lectin CEL-III:: Implications for self-oligomerization and haemolytic processes

CEL-III is a haemolytic lectin, which has two P-trefoil domains (domains 1 and 2) and a P-sheet-rich domain (domain 3). In domain 3 (residues 284-432), there is a hydrophobic region containing two a-helices (118 and 119, residues 317-357) and a loop between them, in which alternate hydrophobic residues, especially Val residues, are present. To elucidate the role of the a-helix region in the haemolytic process, peptides corresponding to different parts of this region were synthesized and characterized. The peptides containing the sequence that corresponded to the loop and second a-helix (119) showed the strongest antibacterial activity for Staphylococcus aureus and Bacillus subtilis through a marked permeabilization of the bacterial cell membrane. The recombinant glutathione S-transferase (GST)-fusion proteins containing domain 3 or the a-helix region peptide formed self-oligomers, whereas mutations in the alternate Val residues in the a-helix region lead to decreased oligomerization ability of the fusion proteins. These results suggest that the a-helix region, particularly its alternate Val residues are important for oligomerization of CEL-III in target cell membranes, which is also required for a subsequent haemolytic action.